Method for inhibiting deoxyribonucleotide triphosphate biosynthesis

ABSTRACT

Process for inhibiting deoxyribonucleotide triphosphate biosynthesis by cells, comprising application onto these cells of at least one of the azo derivatives of the formula (I), in which R 1 , R 2 , R 3  and R 4 =H, Hal, aliphatic or aromatic hydrocarbon or a nitro group, R 1  and R 2  as well as R 3  and R 4  being capable of forming a heterocyclic ring with the N adjacent thereto, X 1  and X 2 =0 or NR 5 , where R 5 =H, Hal, aliphatic or aromatic hydrocarbon, or a nitro group, and the use of these derivatives for the production of medicines to be administered in conditions which require abnormal production of DNA from the cells

This application is a U.S. national phase of PCT/BE97/00104, filed Sep.12, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for inhibitingdeoxyribonucleotide triphosphate biosynthesis by cells, in particularanimal, human or plant cells.

2. The Prior Art

It is known that genetic information is carried by the deoxyribonucleicacid (DNA) present in the cell nucleus. DNA comprises a double helixcomposed of nucleotides, which is fundamental to living organisms,whether animals or plants.

As is known, nucleotides are formed from a sugar, a heterocyclicnitrogenous base and at least one phosphate group. Four phosphates arepresent in the nucleotides which constitute DNA: deoxyguanidinetriphosphate (dGTP), deoxythymidine trisphosphate (dTTP), deoxyadenosinetriphosphate (DATP) and deoxycytidine triphosphate (dCTP).

It was noted some years ago that cancerous cells, because they aredividing rapidly, consume a large quantity of nucleotide triphosphates.Research has consequently focused on medicines capable of inhibiting theformation of the deoxyribonucleotides, such as for example5-fluorouracil, aminopterin and amethopterin (c.f. J. David Rawn,Biochemistry, page 648). Hydroxyurea may also be mentioned, whichnon-selectively inhibits ribonucleotide reductase, and consequently allthe nucleotides involved in DNA synthesis (c.f. “DRUG, Facts andcomparisons”, J.B. Lippincott Company, 1990, pages 2258 and 2259; P.Reichard, “From RNA to DNA, why so many ribonucleotide reductases?”,Science, volume 260, 1993, pages 1773-1776).

The disadvantage of these products is that, given their lack ofselectivity, they inhibit mechanisms which are indispensable to healthy,non-cancerous cells and relate to deoxynucleotides, such as for exampleintracellular energy transport and the enzymatic reactions catalyzedthereby. Treatment with these products consequently entails considerabletoxicity for all cells.

The effect of an analogue of dCTP, cytosine arabinoside or cytarabine,which acts by taking the place of the natural molecule in cellular DNAby means of a competitive phenomenon has already been investigated (c.f.“DRUG” op. cit., pages 2192-2196). This product is not active when takenorally and must be administered with great caution.

Fundamental studies have moreover been undertaken to attempt to combinehydroxyurea with cytarabine. The expected effect was to replace withcytarabine the quantity of dCTP reduced by the action of hydroxyurea(c.f. abstract supplied by the database Medline Express of Schilsky, R.L. et al. “Laboratory and clinical studies of biochemical modulation byhydroxyurea”, Semin. Onc. Jun. 19, 1992 (3, suppl. 9), 84-89).

Cell lines other than those which are cancerous may have a greatlyelevated proliferation rate. Such is the case for lymphocyte cells andthe smooth muscle cells of blood vessels during organ transplants(allografts).

Attempts have already been made to control these cells by restrictingtheir level of deoxynucleotides. Mycophenolic acid (MPA) or one of thederivatives thereof which blocks inosine monophosphate dehydrogenase, sobringing about a reduction in intracellular dGTP and consequentlyblocking DNA synthesis by these cells may be mentioned by way of example(c.f. Pichimayr, R., “Placebo-controlled study of mycophenolate mofetilcombined with cyclosporin and corticosteroids for prevention of acuterejection”, The Lancet, volume 345, May 27, 1995, pages 1321-1325;Sollinger, H. W., “Mycophenolate mofetil for the prevention of acuterejection in primary cadaveric renal allograft recipients”,Transplantation, volume 60, 225-232, number 3, 1995; Gregory, C. R.,“Treatment with rapamycin and mycophenolic acid reduces arterial intimalthickening produced by mechanical injury and allows endothelialreplacement”, Transplantation, volume 59, 655-661, number 5, 1995).

Finally, it is known that viral diseases, in particular AIDS, makesignificant use of the infected cell's genetic material to replicate thevirus. It has recently been discovered that the above-mentionedmycophenolic acid had the ability, given its inhibitory action on theformation of dGTP in cells, to block the activity of reversetranscriptase in vitro and thus to have an anti-HIV effect (V. HiroshiIchimura and J. A. Levy, “Polymerase substrate depletion: A novelstrategy for inhibiting the replication of the human immunodeficiencyvirus”, Virology 211, 554-560, 1995).

The object of the present invention is to provide a process forinhibiting deoxyribonucleotide triphosphate biosynthesis by animal,human or plant cells which does not exhibit the above-stateddisadvantages, in particular unacceptable toxicity for healthy cells,and which thus prevents the large-scale and abnormal cellular productionof deoxyribonucleic acid, which may result, for example, in cancerouscell proliferation.

SUMMARY OF THE INVENTION

This object is achieved by a process as described above, comprisingapplication onto said cells of at least one of the azo derivatives ofthe formula

in which R¹, R², R³ and R⁴ are identical or different and each representa hydrogen or halogen atom or an optionally substituted aliphatic oraromatic hydrocarbon residue, R¹ and R² possibly being connectedtogether to form a heterocyclic nucleus with the nitrogen atom adjacentthereto, and R³ and R⁴ possibly being connected together to form aheterocyclic nucleus with the nitrogen atom adjacent thereto, X¹ and X²are identical or different and each represent an oxygen atom or a groupNR⁵, in which R⁵ is a hydrogen or halogen atom, an optionallysubstituted aliphatic or aromatic hydrocarbon residue, or a nitro group,and in which, when two groups NR⁵ are simultaneously present, each R⁵may be identical to or different from the other, as well as the isomersthereof.

Various of these azo derivatives are known compounds, in particular fortheir antiviral activity, in particular against viruses of theretrovirus group, in particular the AIDS virus (c.f. EP-A-0504184 andEP-A-0524961).

1,1-Azobisformamidine and 1,1′-azobisformamide were prepared as long agoas the end of the last century by J. Thiele (c.f. The Merck Index,10^(th) edition, 919, Rahway, 1983; F. C. Schmelkes et al.,“N,N′-dichloroazocarbonamidine (azochloramide), a N-chloro derivative ofthe oxidant in an oxidation-reduction system”, Journal of AmericanChemical Society, 56, 1610, 1934; FR-B-2056874; U.S. Pat. No. 3,225,026;U.S. Pat. No. 3,684,713). 1,1′-Azobisformamide is known as an additivein flour for food use (U.S. Pat. No. 2,903,361).1,1′-Azobisdimethylformamide has also long been known for itsintracellular oxidising action on the glutathione in human blood cells(N. S. Kosower et al., “Diamide, a new reagent for the intracellularoxidation of glutathione to the disulfide”, Biochemical & BiophysicalResearch Communications, volume 37, number 4, 1969) and for itsinitiation of an additional Ca²⁺ efflux from the liver of perfused rats(H. Sies et al., “Hepatic calcium efflux during cytochromeP-450-dependent drug oxidations at the endoplasmic reticulum in intactliver”, Proc. Natl. Acad. Sci. USA, volume 78, number 6, pages3358-3362). This substance has also been studied for its inhibition ofthe repair of small breaks in DNA strands caused by cell irradiation ina hypoxic environment by means of ionizing radiation (R. E. Meyn et al.,“Post-radiation treatment of CHO cells . . . ”, Radiation Research,volume 94, number 3, 1983, page 614; J. F. Ward et al., “Effects ofinhibitors of DNA strand break repair . . . ”, Cancer Research, 44,1984, pages 59-63). 1,1′-Azobisnitroformamidine has also long been known(W. D. Kumler, “The dipole moments, ultraviolet spectra and structure ofazo-bis-(chloroformamidine) and azo-bis-(nitroformamidine)”, Journal ofAmerican Chemical Society, 75, 3092, 1953). Chloroazodin, which is usedaccording to the invention, has also long been known as a disinfectant(c.f. U.S. Pat. No. 2,073,256 and GB-A-421006).

Observing the effects of these substances on cells in vitro and then inclinical trials did not clarify their mode of action on the HIV viruses.Various investigations have been performed to this end. They led to theconclusion that 1,1′-azobisformamide (ADA) does not inhibit reversetranscriptase in a test system containing no cells at concentrations ofCI₉₀. Simultaneous treatment of MT₄ cells with this substance and HIV-1does not interfere with the integration of proviral DNA. Moreover, ADAdid not seem to inhibit Tat transactivation of gene expression inducedby LTR of HIV-1 (M. Vandevelde et al., “ADA, a potential anti-HIV drug”,AIDS research & human retroviruses, volume 12, number 7, 1996, pages567-568).

This testing has revealed that ADA does not act in the same manner asthe known reverse transcriptase inhibitors conventionally used in AIDStreatment, such as AZT, ddI, ddC and others. It acts at an unidentifiedpost-transcriptional stage. It also does not act in the same manner asthe 7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepine-2(H)-one recentlydeveloped by the company Roche.

Finally, still more recent trials of ADA have demonstrated that ADA hadno protease inhibiting effect at the post-transcriptional stage and thusdid not act in the same manner as the protease inhibitors recently usedin treating AIDS.

Despite these successive failures to understand the mode of action ofADA on the HIV viruses, a new trial has been set under way to determinewhether this substance does not act on the treated cells in the samemanner as hydroxyurea. This latter substance is formed from a moleculedistantly related to ADA, although it is not an azo derivative.

This comparative trial is described in greater detail in the Examplesbelow.

It would appear from this trial that, on the one hand, ADA does indeedact on the biosynthesis of cellular deoxyribonucleotide triphosphates,which explains its posttranscriptional mode of action on the HIVviruses, but that, on the other, its action differs entirely from thatof hydroxyurea. ADA is in fact not a non-selective ribonucleotidereductase inhibitor, but is on the contrary a derivative which actsprimordially to inhibit the formation of dCTP present in cells. Itshould be noted that deoxycytidine triphosphates comprise the group ofdeoxynucleotide phosphates having the lowest concentration in the cells.A reduction in the content in or selective disappearance of thesedeoxycytidine triphosphates from the cell thus prevents the cell frombiosynthesizing DNA, while requiring for this purpose a substantiallylower active substance concentration than that required by hydroxyureato inhibit all the deoxynucleotides.

Biosynthesis inhibition, which in the case of ADA results inpreferential inhibition of the formation of deoxycytidine triphosphates,could proceed as a consequence of ADA acting on different cellularenzymatic targets. It could be predicted that ADA would have aninhibitory action on UMP/CMP kinase (uracil monophosphate/cytidinemonophosphate kinase), which would block the formation of deoxycytidinediphosphate (dCDP) from deoxycytidine monophosphate (dCMP), as well asthe formation of cytidine diphosphate (CDP) from cytidine monophosphate(CMP). ADA could thus be viewed as having an effect on cytidinetriphosphate (CTP) synthetase, which would block the formation of thissubstance from uracil triphosphate (UTP). Other targets could also beconsidered, such as for example inter alia NDP kinase, although theseare less probable in the specific case of ADA.

Moreover, it should be noted that ADA has long been known to benon-toxic to human beings (c.f. B. L. Oser et al., “Studies of thesafety of azodicarbonamide as a flour-maturing agent”, Toxicology &Applied Pharmacology, 7, 445-472, 1965).

Furthermore, a trial has already been conducted on healthy volunteerswho were treated for 30 days with 1500 mg per day of ADA, in three 500mg of doses, without any side-effects (c.f. EP-0524961, Example 12).Clinical trials were then undertaken. Ten volunteers took ADA for 3months at dosages of 1 g three times daily for the first month, 2 gthree times daily for the second month and 3 g three times daily for thethird month. No serious side-effects were observed, apart from oneepisode of nephritic lithiasis at the 9 g/d dose in one of the patientsas a result of a build-up of the catabolite of the product (biurea) inthe kidneys.

It is also known that when 1,1′-azobisdimethylformamide is used in thetreatment of healthy cells, it has no toxic effect on the cells even atrelatively high concentrations (c.f. EP-A-0524961, Examples 9a) and10a)). This same effect has been observed for1,1′-azobisdimethylformamidine (c.f. EP-A-0524961, Example 8a)) and for1,1′-azobisformamide (c.f. EP-A-0524961, Example 11a)).

Finally, it is also known that, at concentrations of 660 μg/ml,chloroazodin does not reduce the viability of healthy cells and does notreach the environmental toxicity threshold as determined by acute fishtoxicity (c.f. EP-A-0504184, Examples 6 and 12).

It may thus be concluded that the derivatives to be used according tothe invention not only allow an action to be exerted upondeoxyribonucleotide triphosphate biosynthesis in the treated cells atrelatively low doses, but moreover have very low intrinsic toxicitytowards the human body or the healthy cells which are treated.

According to one embodiment of the invention, at least one of the statedazo derivatives is applied onto cells isolated from macroorganisms oronto cells of microorganisms, for example originating from cellcultures. According to the invention, it is also possible to envisagesuch application onto the cells of an organism or multicellular tissueextracted from a human or animal body, for example onto blood vesselcells or cells from a sample of blood or lymph, as well as onto thecells of a graft which is to be introduced after said application intothe body of a human or animal, for example a heart or kidney. A graftmay be taken to mean an allograft to be introduced into the body ofanother human or animal and the extraction of the organism ormulticellular tissue may be taken to be definitive. Phytosanitarytreatment of plants, for example of seeds or developed plants, may alsobe considered for treatment with these azo derivatives.

The invention also relates to the use of at least one azo derivative asstated above for the production of medicines for use in the treatment orprevention of human or animal conditions which give rise to large-scaleand abnormal cellular production of deoxyribonucleic acid, with theexception of viral diseases, in particular infections by viruses of theretrovirus group. Antineoplastic medicines may in particular beprovided, such as medicines to combat liquid and solid tumors, such asantileukemic and antitumor medicines.

The invention also provides a method for therapeutic or preventivetreatment of a human or animal body possibly exhibiting large-scale orabnormal cellular production of deoxyribonucleic acid, with theexception of those affected by a viral disease, in particular by aninfection with a virus of the retrovirus group, wherein this methodcomprises the administration to said human or animal body of atherapeutically effective quantity of an active substance selected fromamong one or more of the azo derivatives of the above-stated formula.

The invention will now be described in greater detail by means of thefollowing, non-limiting Examples.

EXAMPLE 1 Comparative Trial Between ADA and Hydroxyurea

This trial was conducted by the Antiviral Research Laboratory of theCenter for Drug Evaluation & Research of the Food & Drug Administrationof the United States of America.

Method:

Cells.

Human A3.01 cells were incubated with 0, 10, 100, 200 micromolarconcentrations of ADA and with a 100 micromolar concentration ofhydroxyurea in RPMI 1640 with the addition of 10% of fetal calf serum, 4mM of L-glutamine, in a humidified atmosphere of 95% air and 5% CO₂.

Cells undergoing logarithmic growth were used for the determinations ofdeoxynucleotide triphosphates (DNTP) and ribonucleotide triphosphates(rNTP).

Preparation of Cell Extracts for HPLC

After incubation, 60% methanolic extracts were prepared fromheat-inactivated A3.01 cells and analyzed as described in Ford, H. Jr.et al., Cancer Research, 51, 3733-3740, 1991.

Determination of intracellular rNTP and dNTP pools by HPLC ion exchangegradient.

The cellular ribonucleotides were measured by ion exchange HPLC onPartisil 10 Sax columns as described in Ford et al., op. cit.

The deoxyribonucleotides were determined using sodium periodate in orderto eliminate the ribonucleotides; the deoxyribonucleotides were thendetermined by ion exchange HPLC as described in Hao, Z. et al., Mol.Pharmacol., 34, 431-435, 1988.

Results

Effect of ADA on rNTP pools in A3.01 cells

Effect of ADA on rNTP pools in A3.01 cells UTP* (nmole/10^(4 cells) CTP*ATP* GTP* ADA (10⁻⁶ M)  0 0.71 0.24 2.51 0.38  10 0.87 0.28 2.85 0.45100 1.10 0.31 2.64 0.47 200 0.49 0.09 1.31 0.22 Hydroxyurea (10⁻⁶ M) 1001.01 0.34 3.69 0.93 Effect of ADA on dNTP pools in A3.01 cells dTTP(pmole/10⁶ cells) dCTP dATP dGTP ADA (10⁻⁶ M)  0 39.7 12.7 50.3 17.2  1039.2 6.5 53.6 19.4 100 40.3 6.2 52.1 17.1 200 19.1 2.9 22.8 15.1Hydroxyurea (10⁻⁶ M) 100 21.6 3.6 9.0 19.3 *UTP = uracil triphosphate;CTP = cytosine triphosphate; ATP = adenosine triphosphate; GTP =guanosine triphosphate.

The azodicarbonamide compound thus exhibits a preferential inhibitoryeffect on the dCTP pool (50% inhibition at concentrations of 10 μM and100 μM).

A higher concentrations, ADA exhibits a more cytotoxic effect, which isevident from the reduction in the rNTP pools and dNTP pools.

Hydroxyurea, a known ribonucleotide reductase inhibitor, is verydifferent from ADA. It reduces the pools of DATP, dCTP and dTTP, whileincreasing rNTP pools at a concentration of 100 microM. The rNTP/dNTPratio in cells treated with hydroxyurea (68.2) (reduction in dNTP poolsand increase in rNTP pools) in comparison with that observed inuntreated cells (24.33), indicates that hydroxyurea is a specificribonucleotide reductase inhibitor, while the effect of ADA ispreferentially specific to the CTP/dCTP ratio.

EXAMPLE 2

The preferential effect of ADA on the intracellular dCTP pools observedin Example 1 has been confirmed by further investigations conducted bythe Antiviral Research Laboratory of the Center for Drug Evaluation &Research of the Food & Drug Administration of the United States ofAmerica.

Method:

Peripheral human blood cells from healthy donors (peripheral bloodmonocytic cells=PBMC) are stimulated by phytohemagglutinin (PHA) and arethen incubated with concentrations of 0, 50, 100, 200 μmoles of ADA inan appropriate culture medium. The cell extracts were investigated usingthree different methods.

1) High performance liquid phase chromatography (HPLC) of the completeextract.

2) HPLC analysis of the extracts after destruction of the precursors(uridine triphosphate, cytidine triphosphate, adenosine triphosphate)with periodate.

3) Analysis by a method after F. Poder comprising a determination bydetection using specific DNA probes and amplification.

The results are shown in Table 1 below.

TABLE 1 Preferential effect of ADA on dCTP pools in PHA-stimulated PBMCHPLC Peridate F. Poder Mean Percentage Method dCTP dCTP dCTP dCTPinhibition Quantities of ADA (μM)  0 3.01 1.42 1.76 2.06 0  50 0.09 1.160.88 0.71 66 100 0.07 0.76 0.61 0.48 77 200 0.05 0.6 0.46 0.37 82

EXAMPLE 3

Investigation of SUP-T1 cells (continuous line of human lymphoblasticcells) which were stimulated by PHA and then treated by doses of 10 μgand 20 μg of ADA/ml of culture medium.

The number of cells was standardized to 5·10⁴ cells per ml at day 0.Inhibition of proliferation is expressed as the percentage of theproliferation of the control.

Three evaluation methods were used at the Biochemistry & NutritionLaboratory of the Université Libre de Bruxelles and at this university'sImmunology Laboratory.

Incorporation of tritiated thymidine into the cells as a radiographicmarker (experiments repeated 8 times).

Colorimetric marking of cell death by MTT (experiments repeated 12times).

Trypan blue exclusion method, which is an indicator of cell membraneintegrity (experiments repeated 8 times).

The results are shown in Table 2 below. There is a distinct effect onthe proliferation of a continuous human cell line.

TABLE 2 Inhibition of proliferation of SUP-T1 cells (examination after48 hours) ADA ADA Method 20 μg/ml 10 μg/ml 3H═thymidine 18 h 39% ± 1815% ± 6  MTT 43% ± 8  19% ± 10 Trypan blue 37% ± 17 24% ± 10

EXAMPLE 4 Capsules for Oral Administration

Composition of One Capsule:

500 mg of ADA

10 mg of glycerine monostearate

10 mg of precipitated silicon dioxide

5 mg of magnesium stearate.

This composition is packaged in gelatine capsules in a conventionalmanner. It is, for example, possible to administer two capsules threetimes daily to patients exhibiting symptoms of pathological cellproliferation. The capsules may be administered alone or in combinationwith any other appropriate antiproliferative treatment.

EXAMPLE 5 Coated Tablets

Tablets containing 250 mg of azobisformamidine were produced in aconventional manner using the following excipients: hydroxypropylmethylcellulose, hydroxypropyl cellulose, titanium dioxide, polyethyleneglycol 400, black iron oxide.

These tablets may be administered to bring about an adequate reductionin intralymphocytic deoxycytidine triphosphates (determination by HPCafter isolation of lymphocytes).

This treatment will be instituted to induce and maintain remission inacute lymphatic leukemia (lymphoblastic or lymphocytic leukemia) or inmyeloblastic leukemia. The active substance is administered alone orpreferably in combination with mercaptopurine or azathioprine or anyother appropriate drug.

EXAMPLE 6 Injectable Form

An injectable form is prepared from 1 g of dimethylazobisformamide andapyrogenic distilled water with added NaCl.

These forms should be administered, for example, in cases ofadenosarcoma, especially generalized adenosarcoma.

This treatment may be combined with azathioprine, 5-fluorouracil orother conventional treatments.

EXAMPLE 7 Cream or Ointment

A cream or ointment is prepared from azobis[chloroformamidine](chloroazodin) and, as excipient, in particular glycerine, paraffin oil,petrolatum.

This cream may be applied topically as a supportive treatment for skincancers, for example epidermoid epithelioma.

EXAMPLE 8

Transdermal administration systems for dimethylazobisformamide may alsobe provided.

These systems may be administered in order to achieve a sustainedreduction in intracellular dCTP in patients suffering from Hodgkin's ornon-Hodgkin's lymphoma.

This treatment may be combined with injected cytarabine.

It must be understood that the present invention is not in any waylimited to the methods and embodiments stated above and thatconsiderable modifications may be made without extending beyond thescope of the attached claims.

What is claimed is:
 1. Process for inhibiting deoxyribonucleotidetriphosphate biosynthesis by cells, comprising application onto saidcells of at least one of the azo derivatives of the formula

in which R¹, R², R³ and R⁴ are identical or different and each representa hydrogen or halogen atom or an optionally substituted aliphatic oraromatic hydrocarbon residue, R¹ and R² possibly being connectedtogether to form a heterocyclic nucleus with the nitrogen atom adjacentthereto, and R³ and R⁴ possibly being connected together to form aheterocyclic nucleus with the nitrogen atom adjacent thereto, X¹ and X²are identical or different and each represent an oxygen atom or a groupNR⁵, in which R⁵ is a hydrogen or halogen atom, an optionallysubstituted aliphatic or aromatic hydrocarbon residue, or a nitro group,and in which, when two groups NR⁵ are simultaneously present, each R⁵may be identical to or different from the other, as well as the isomersthereof.
 2. Process according to claim 1, comprising inhibition of theformation of at least one deoxyribonucleotide triphosphate.
 3. Processaccording to claim 2, comprising inhibition of the formation ofdeoxycytidine triphosphate during said biosynthesis.
 4. Processaccording to claim 1, wherein R¹ to R⁵ each represent an aliphatic oraromatic hydrocarbon residue comprising from 1 to 6 carbon atoms. 5.Process according to claim 1, wherein the azo derivative is selectedfrom among the group comprising derivatives of azobisformamidine,derivatives of azobisformamide, and 1,1′-(azodicarbonyl)-dipiperidine.6. Process according to claim 1, including the step of applying the azoderivative onto cells at a 10 to 200 micromolar concentration. 7.Process according to claim 6, including the step of applying the azoderivative onto cells at a 10 to 100 micromolar concentration. 8.Process according to claim 1, including the step of applying acomposition comprising at least one of the said azo derivatives and anappropriate excipient.
 9. Process according to claim 1, including thestep of applying said azo derivatives onto cells isolated frommacroorganisms or onto cells of microorganisms.
 10. Process according toclaim 1, including the step of applying said azo derivatives onto cellsof an organism or multicellular tissue extracted from a human or animalbody.
 11. Process according to claim 10, wherein the organism ormulticellular tissue is a graft.
 12. Process according to claim 1including the step of applying said at least one azo derivative ontoplants.
 13. Process according to claim 1, including the step of applyingsaid at least one azo derivative onto animals cells.
 14. Processaccording to claim 1, including the step of applying said at least oneazo derivative onto humans cells.
 15. Process according to claim 1,including the step of applying said at least one azo derivative ontoplant cells.
 16. Process according to claim 5, wherein the derivativesof azobisformamidine are selected from the group consisting of1,1′-azobisformamidine, 1,1′-azobisnitroformamidine,2,2′-azobismethylformamidine, 1,1′-azobisfluoroformamidine,1-monochloro-azobisformamidine and azobis.
 17. Process according toclaim 5, wherein the derivatives of azobisformamide are selected fromthe group consisting of 1,1′-azobisformamide anddimethylazobisformamide.
 18. Method for therapeutic treatment of a humanor animal body exhibiting abnormal cellular production ofdeoxyribonucleic acid, with the exception of those affected by a viraldisease, comprising the stage of administration of a therapeuticallyeffective quantity of an active substance to said human or animal body,this active substance being selected from among one or more of the azoderivatives of the formula

in which R¹, R², R³ and R⁴ are identical or different and eachrepresents a hydrogen or halogen atom or an optionally substitutedaliphatic or aromatic hydrocarbon residue, R¹ and R² possibly beingconnected together to form a heterocyclic nucleus with the nitrogen atomadjacent thereto, and R³ and R⁴ possibly being connected together toform a heterocyclic nucleus with the nitrogen atom adjacent thereto, X¹and X² are identical or different and each represents an oxygen atom ora group NR⁵, in which R⁵ is a hydrogen or halogen atom, an optionallysubstituted aliphatic or aromatic hydrocarbon residue, or a nitro group,and in which, when two groups NR⁵ are simultaneously present, each R⁵may be identical to or different from the other, as well as from amongthe isomers thereof.
 19. Method for therapeutic treatment according toclaim 18, wherein said abnormal cellular production is a leukemia or atumor.